Natural products are the source of the majority of FDA approved drugs and, in particular, metabolites produced by microorganisms are the source of many of the antibiotics and anticancer agents used today. Over the past two decades large screening programs have largely deemphasized microbial extracts in spite of the historical success of microbial natural products as lead structures for the development of small molecule therapeutics and the continuing clinical need for new antimicrobials and chemotherapeutics. It is estimated that less than one percent of the bacteria present in the environment is readily cultured in the laboratory and therefore accessible to use in traditional small molecule discovery strategies. Uncultured bacteria are likely the largest remaining pool of biosynthetic diversity that has not yet been examined for the production of bioactive natural products. While it is still not possible to easily culture most bacteria in the environment, it is possible to extract microbial DNA directly from environmental samples (environmental DNA, eDNA) and clone this DNA into easily cultured bacteria where, for the first time, it can be functionally characterized. In this study a collection of eDNA mega-libraries constructed from soils collected in the four major deserts of the American Southwest (Sonoran Desert, Chihuahuan Desert, Mojave Desert and the Great Basin Desert) will be constructed and arrayed to permit screening for novel natural product biosynthetic gene clusters. Two related but complementary DNA-based screening approaches will then be used to recover novel small molecule biosynthetic gene clusters from these eDNA mega-libraries. In the first approach we will focus on the identification and recovery of clones containing biosynthetic systems with a history of producing structurally diverse bioactive natural products. In the second strategy we will focus on the identification and recovery of clones containing new variants of specific classes of pharmacologically important natural products. Molecules encoded by new environmental DNA-derived gene clusters will then be functionally accessed using heterologous expression in phylogenetically diverse cultured bacteria, and these metabolites will be tested in whole cell antibacterial, antifungal and anticancer assays. The historical success of natural products as starting points for the discovery of therapeutics provides support for our hypothesis that the metabolites found in these studies will have a high potential of being beneficial to human health. PUBLIC HEALTH RELEVANCE: Bacteria that have not yet been cultured outnumber their cultured counterparts by at least two to three orders of magnitude and are likely to be a rewarding source of new biologically active small molecules. The proposed systematic examination of the biosynthetic capacity present in naturally occurring bacterial populations, instead of just the small fraction of bacteria that are easily cultured in the laboratory, should increase the number and diversity of natural products that are available to test as lead structures for the development of small molecule therapeutics.